Tools for the Characterization of Biomass at the Nanometer Scale - The Nanoscience and Technology of Renewable Biomaterials - page 67

the entire structure. The structure of cellulose, the various crystalline forms, and the
issue of crystallinity are discussed in Chapter 6.
Spectroscopic methods of measuring cellulose crystallinity (NMR, Raman, IR) are
typically used with pure cellulose samples because hemicellulose and amorphous cellu-
lose produce very similar signals, whereas lignin is much different. The X-ray diffraction
method, by contrast, can be used on native biomass samples because the lignin, hemi-
cellulose, and amorphous cellulose all contribute to a broad background peak, whereas
the cellulose crystals produce a sharper peak.

3.3.2.1 X-ray

Cellulose crystallinity index (CrI), or percentage by mass of crystalline cellulose in
a sample, is determined by diffracting or reflecting Cu K α X-rays off of randomly
oriented, relatively pure cellulose specimens. The original method is still widely used.
In this method, the peak intensity of the 002 peak (I 002 ,2 = 22 . 6 ◦ ) diffracted from

the crystalline cellulose is compared with the intensity of the reflection from amorphous
cellulose, (I amorph ) , which has a very broad peak at 18 ◦ (44).

I 002 − I amorph

I 002

CrI = × 100 (3.7)

Segal (44) also discusses the effect of specimen packing, density, orientation, and other
factors that all must be controlled to get reproducible results.
Crystal width can be calculated using the Scherrer equation on the peak assigned to
(002) planes:

D = Kλ(B cos )

where D is crystal thickness, λ the radiation wavelength, the diffraction angle, and B
the full width of the diffraction peak measured at half-maximum height. The correction
factor, K , is typically set at 0.9 (45).
Because of the small crystal size and subsequent broad peaks, numerous efforts have
been made to improve quantification. These range from simply taking areas rather
than heights for the crystalline and amorphous reflections (46) to modeling the entire
diffraction pattern (47). If treatments partially dissolve cellulose, then different crystal
forms, such as cellulose II, and their reflections are likely to appear upon solvent removal.
The exact peak positions are reported to vary by more than 0 . 5 ◦ .

3.3.2.2 13 CNMR
Solid-state 13 C NMR is often used to assay the amount of cellulose in crystalline vs.
amorphous form. The anhydroglucose carbon 4 (C4) peak is shifted 89 ppm when
inside the crystal and 86 ppm when on the crystal surface or in amorphous cellulose
and hemicellulose (45, 48). In highly crystalline specimens, the C6 peak at 65 ppm
can be compared with the amorphous peak at 60-62 ppm. Band assignments have
been made for all the components (49), and the effect of some factors such as residual
hemicellulose and crystal polymorph (I α vs. I β ) on the observed crystallinity has been
described (50, 51).